This document has been prepared with financial support from the Commission of the European Communities, specific RTD programme in the Agriculture and Fisheries FAIRCT98-4094, a concerted action called Quality control measures in the production and processing chain to reduce Fusarium mycotoxin contamination of food and feed grains.

Contributing authorsA. De Girolamo Institute of Sciences of Food Production (CNR-ISPA), Via Einaudi 51, Bari, 70125 Italy T.W. Hollins Monsanto UK Ltd. The Maris Centre, Hauxton Road, Trumpington, Cambridge, CB2 2LQ, United Kingdom P. Jennings Central Science Laboratory Sand Hutton, York, Yorkshire, Y04 1LZ United Kingdom H.C. de Jong Cebeco Seeds B.V. P.O. Box 139, 8200 AA Lelystad, The Netherlands D.G. Kloet State Institute for Quality Control of Agricultural Products (RIKILT) P.O. Box 230, Bornsesteeg 45, 6700 AE Wageningen, The Netherlands J. Khl Plant Research International P.O. Box 16, 6700 AA Wageningen, The Netherlands G. Koornneef Dutch Board for Arable Crop Production P.O. Box 29739, 2502 LS 's Gravenhage, The Netherlands E. Meekes Plant Research International P.O. Box 16, 6700 AA Wageningen, The Netherlands T. Miedaner University of Hohenheim State Plant Breeding Institute (720), Fruwirthstr. 21, D-70593 Stuttgart, Germany A.P.M. den Nijs Plant Research International P.O. Box 16, 6700 AA Wageningen, The Netherlands W.A. van Osenbruggen TNO Nutrition and Food Research P.O. Box 360, 3700 AJ Zeist, The Netherlands H. Pettersson Swedish University of Agricultural Sciences (SLU). Department of Animal Nutrition and Management, Animal Husbandry P.O. Box 7024, Uppsala, 750 07 Sweden P. Ruckenbauer Institute for Agrobiotechnology (IFA-Tulln) Konrad Lorenz-Strasse 20, Tulln, A-3430 Austria O.E. Scholten Plant Research International P.O. Box 16, 6700 AA Wageningen, The Netherlands A. Visconti Institute of Sciences of Food Production (CNR-ISPA), Via Einaudi 51, Bari, 70125 Italy

ContentsPreface Acknowledgements Summary 1 1. The Concerted Action Mycotochain: an introduction 5 1.1 Objectives 5 1.2 Research Tasks 5 2. Crop production 7 2.1 Introduction 7 2.2 Breeding for resistance to Fusarium spp. in wheat 7 2.3 Ring test with selected European winter wheat varieties 17 2.4 Control of the fungus through the use of fungicides 22 2.5 Risk factors in Fusarium head blight epidemics 26 3. Fusarium mycotoxins in cereals: storage, processing and decontamination 30 3.1 Introduction 30 3.2 Fungal and mycotoxin contamination in stored crops 31 3.3 Food processing and detoxification of Fusarium mycotoxins 32 3.4 Recommendations and future research 40 4. Analysis of relevant Fusarium mycotoxins in cereals - the state of the art 42 4.1 Introduction 42 4.2 Fusarium mycotoxins - analysis 43 4.3 Recommendations and future research 48 5. Tools to improve food safety in the chain 52 5.1 Introduction 52 5.2 Risk analysis 52 5.3 HACCP principles as a tool in the prevention of Fusarium mycotoxins in the cereal chain 54 5.4 Good Agricultural Practice and EUREPGAP 59 5.5 Legal limits for Fusarium mycotoxins in the EU 60 5.6 Harmonisation of standards for mycotoxins in the Codex Alimentarius 63 6. The Chain-wide approach: Final conclusions and needs for further research 66 6.1 Future breeding and research needs in the EU 66 6.2 Final recommendations for the chain-wide approach 66 References 70 Appendix I. Partners of Mycotochain - 8 pp. Appendix II. European Research Projects related to Mycotoxins in cereals - 2 pp.

PrefaceThe document Food safety of cereals: A chain wide approach to reduce Fusarium mycotoxins is the final deliverable of EU FAIR-CT98-4094, i.e. the Concerted Action Quality Control Measures in the Production and Processing Chain to Reduce Fusarium Mycotoxin Contamination of Food and Feed Grains with the acronym Mycotochain. The Concerted Action started in January 1999 and involved three well attended general meetings of all participants from eight European countries: Austria, Denmark, France, Germany, Italy, Sweden, the Netherlands and the United Kingdom. Partners belong to fundamental research organisations, such as universities and governmental institutes, commercial breeding companies, the trade, a millers association, a milling company and food and feed safety organisations. The number of partners has steadily grown throughout the course of the project. The meetings led to extensive exchange of information between chain partners and young scientists took part in exchange and mobility programs as part of this Concerted Action. The Concerted Action has divided its activities in the following task groups: Reduction of Mycotoxin Contamination during Crop Production Reduction of Mycotoxin Contamination during Storage and Processing of Grain Improved Methodology to Measure Mycotoxin Contamination Integrated Chain Wide Approach For each task group a chairman was appointed. This document presents an overview regarding Fusarium research and mycotoxin contamination of cereals, mainly wheat, but also maize, the effect of processing and possibilities of decontamination of cereals and the state of the art of methodologies to measure mycotoxins in the chain. The document is the result of collaboration between European partners, which represent all parts of the production chain of cereals and cereal based products and can be regarded as an integrated chain wide approach. The task group chairmen have played a major role in assembling the present document. They brought up information about their specific activities. Apart from the task group activities, information is presented about risk analysis, HACCP, legislation and EUREPGAP, followed by a final chapter with conclusions and recommendations for further research to be financed by the Sixth Framework Programme of the European Union. During the course of the project, we have felt an increasing awareness of the mycotoxin problem amongst the participants and growing sense of urgency to invest in solutions for this threat to our food and feed chain. Therefore several participants have contributed to an Expression of Interest regarding mycotoxin control. We trust that this document may be useful as means to re-affirm the links between the partners in the cereal chains, so that the ultimate goal: mycotoxin-free cereals and cereal products, may become a reality to the benefit of European consumers. June 2002, A.P.M. (Ton) den Nijs & Olga E. Scholten (chairman and secretary of Mycotochain) Note: also visit our internet site, created especially for the project http://www.mycotochain.org

AcknowledgementsThe Concerted Action Quality Control Measures in the Production and Processing Chain to Reduce Fusarium Mycotoxin contamination of food and feed grains, abbreviated as Mycotochain, contract number FAIR-CT98-4094, has been carried out with financial support of the European Union under Framework Programme 5. We are grateful for the opportunity the grant provided for meetings of authors from the participating groups and for financial support for the printing of this document. We express our appreciation to all partners for their involvement in the project, and our sincere gratitude to the task group chairmen, Peter Ruckenbauer of Austria, Ton van Osenbruggen of the Netherlands and Angelo Visconti of Italy, because without their enthousiastic input this document would not have been printed. We also thank Annalisa de Girolamo, Bill Hollins, Phillip Jennings, Hein de Jong, David Kloet, Jrgen Khl, Gerrit Koornneef, Ellis Meekes, Thomas Miedaner and Hans Pettersson for their contributions in this document. Furthermore, we thank Hans de Keijzer for critical reading of the manuscript and Monica Olsen for her information on HACCP. Piet Boonekamp, Ruud van den Bulk, Gert Kema, Huub Lffler and Cees Waalwijk of Plant Research International, the Netherlands, are acknowledged for discussions regarding Framework Programme 6 and Marian van Harmelen for her help compiling the document.

SummaryFusarium head blight (FHB) in wheat and barley and Fusarium ear rot in maize is caused by several Fusarium species. Infection with Fusarium fungi firstly decreases the yield of the crop due to the production of shrunken kernels. More importantly, however, the disease reduces the quality of the seed since several of these fungi produce mycotoxins. Examples of mycotoxins produced by Fusarium in cereals are deoxynivalenol, nivalenol, fumonisins and zearalenone. From a food safety point of view, consumption of mycotoxin-infected cereals is dangerous as it threatens the health of men and animals. Currently, the EU is working on legislation and set maximum tolerated levels of mycotoxin concentrations in flour and cereal products. This working document Food safety of cereals: A chain wide approach to reduce Fusarium mycotoxins is the final deliverable of EU FAIR-CT98-4094, i.e. the Concerted Action Quality Control Measures in the Production and Processing Chain to Reduce Fusarium Mycotoxin Contamination of Food and Feed Grains with the acronym Mycotochain. The Concerted Action has brought together over 20 European actors in the cereals production chain during the period January 1999 through June 2002, to discuss the possibilities to reduce Fusarium mycotoxin contamination of food and feed grains. Partners originate from eight European countries: Austria, Denmark, France, Germany, Italy, Sweden, the Netherlands and the United Kingdom and belong to fundamental research organisations, such as universities and governmental institutes, commercial breeding companies, the trade, a millers association, a milling company and food and feed safety organisations. This document describes the activities of the Concerted Action, which was structured in four task groups. An Introduction to the Concerted Action is presented in chapter 1. An out-line is given of the objectives as well as of the research tasks. The main objective of this Concerted Action is the exchange of knowledge between partners. To stimulate this exchange of knowledge three general meetings were organised in which partners were invited to present their research results, to share information regarding the Fusarium-mycotoxin problem and to discuss relevant topics in the mycotoxin field. Exchange of information between partners resulted in the writing of the chapters 2, 3 and 4 of this document. Chapter 2 deals with various aspects of crop production. The first part of this chapter informs about breeding for resistance in wheat. A description is given of the Fusarium species that are involved in the infection process and the types of mycotoxins that are being produced. The problem of mycotoxin contamination is a problem that starts at the beginning of the production chain where susceptible wheat varieties are used. The use of resistant varieties is important to reduce mycotoxin contamination. Since, however, no 1

high-yielding resistant varieties exist, breeding for resistance is necessary. Breeding for resistance is important to reduce mycotoxin contamination. Resistance inherits mostly dominantly, but is controlled by a number of genes. Molecular markers seem interesting to accelerate the breeding process. Based on experiments, it is expected that resistance to Fusarium is quite durable. The same chapter also informs about the results of a ring test carried out by three partners involved in breeding. In this test, 17 varieties were screened for resistance to FHB on 5 locations in Europe of which three were artificially infected and the other two naturally. In general, disease estimates for varieties ranked similarly at different sites. Although some varieties were only slightly infected, the results clearly showed that none of them was completely resistant. The correlation between disease incidence and DON content for these samples was estimated as 0.80. Fungicides may be used to control the disease. So far, however, the effect of fungicides has been inconsistent. Product choice and timing of application as well as rate of application are important factors to keep in mind to achieve optimal control of FHB. In chapter 3 an overview is presented of problems occurring after harvesting the grain: during storage, processing and decontamination. The humidity and the temperature are important factors that influence fungal growth. During storage also damaging of seeds may result in higher contamination, especially in maize. Food processing that may involve physical and/or chemical decontamination could be considered as a strategy to destroy mycotoxins. The ideal decontamination procedure should be easy to use, inexpensive and should not lead to the formation of compounds that are still toxic or can alter the nutritional and palatability properties of the grain or grain products. Strategies for intentional detoxification or decontamination of commodities containing mycotoxins can be classified as chemical, microbiological or physical and are explained in detail. It is concluded that more research is needed to further optimise decontamination procedures. The state of the art regarding the analysis of relevant Fusarium mycotoxins in cereals is described in chapter 4. There is a need for good and standardised analytical methods to make surveys and control of these toxins possible, but in most cases they are still lacking. The methods for many of the toxins are complicated and often lead to high variations both within and between laboratories. Activities supported by the European Commission are going on to improve the analytical methods. Results from such studies and others are presented. For trichothecenes (e.g. deoxynivalenol, nivalenol) HPLC has been used as well as capillary gas chromatographic methods with EC or MS-detection, which are preferred for their higher sensitivity and selectivity. Also methods for rapid screening, such as ELISA and colorimetric bioassays are discussed. For zearalenone, although TLC, GC and GC-MS methods are available, HPLC is mostly used. For fumonisins, a validated HPLC method has been developed that meets CEN criteria. The chapter ends with recommendations for further optimisation of LC-MS(-MS), near infra red transmittance and biosensors.

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In the meantime in Europe and the USA, project partners were aware of or played a role in many other activities concerning the problem of mycotoxin contamination in cereals, such as e.g. HACCP, good agricultural practice, legislation, the Codex Alimentarius. These activities have been compiled in chapter 5. To apply HACCP to establish minimum mycotoxin contamination, the following prerequisites should be taken into account: good hygiene practice, good agricultural practice, good storage practice, good manufacturing practice, management/stake holder commitment as well as training. Recommendations to improve good agricultural practice during the whole cereal production chain are presented. Under legislation EU opinions on Fusarium mycotoxins is listed together with their internet addresses. The final chapter 6 is the result of a discussion among the co-ordinator, the task group chairmen and the secretary. It identifies the needs for further concerted research and breeding and gives recommendations for the chain-wide approach. This evolves into the expression of interest regarding the mycotoxin control problem in which several of the participants of the concerted action participate. The group has not drawn up a protocol for use throughout the chain, since organisations such as EUREPGAP and the Codex Alimentarius, have already studies underway for establishing such protocols. The data in this report may be used to underpin such protocols as needed.

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1. The Concerted Action Mycotochain: an introduction1.1 ObjectivesFusarium fungi are an important problem in the cereal food and feed chain because of their ability to produce mycotoxins in the grain, which cause serious illness and immunorepression in humans and animals, as well as yield loss per se. This mycotoxin problem is the result of events at the start of the chain, due to fungal infestation during the growing of the cereal crop, while the negative effects are found at the end of the chain, in food and feed products. The control of this problem therefore calls for collaboration throughout the chain of production and processing. The long term objective of this Concerted Action was to establish a working relationship between European partners of the production chain of cereal based food and feed products to be able to prevent mycotoxin contamination due to Fusarium fungi (see Appendix I for an overview of the partners). The concerted action started in January 1999 with a general meeting and ran through 2001. It was extended with an extra half year in order to produce the final document with the Taskgroup chairmen and ended July 1, 2002. Within the timeframe of this Concerted Action we achieved Exchange of knowledge on problems caused by infection due to Fusarium fungi throughout the production chain of cereals Identification of possibilities for effective collaboration to minimise mycotoxin contamination of cereal based products Identification of suitable quality control opportunities for mycotoxin content throughout production and manufacturing of food and feed products Establishment of opportunities for future collaboration Identification of gaps in knowledge which require further research

1.2 Research TasksWithin this Concerted Action partners were brought together that represent all parts of the production chain of cereals and cereal based products. Initially the project was divided into five Task groups, which concentrated on various parts of the production chain:

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Task 1. Reduction of mycotoxin contamination during Crop Production Task 2. Reduction of mycotoxin contamination during Transportation and Storage Task 3. Reduction of mycotoxin contamination during Processing of grain Task 4. Improved methodology to Measure trichothecene and fumonisin Task 5. Integrated Chain Wide Approach

During the project phase, it became clear that Task 2 overlapped with other Tasks. Therefore, it was concluded to leave out Task 2 and consider the tasks like investigation of cereal samples at the farms for Task 1, the storage for Task 3 and the methodology needed to investigate samples during transportation and storage for Task 4. Within these specific task groups the various partners exchanged results of their own activities first, and subsequently interacted with other task groups to achieve improved quality throughout the production chain. The project has resulted in three well-attended general meetings where scientific and practically oriented partners discussed freely and enthusiastically about possible solutions for the problems encountered. Exchange of information among partners resulted in the writing of the chapters 2, 3 and 4 of this document. In the meantime in Europe and the USA, project partners were aware of or played a role in many other activities concerning the problem of mycotoxin contamination in cereals, such as e.g. HACCP, good agricultural practice, legislation, the Codex Alimentarius, which have been compiled in chapter 4 (see also Appendix II for an overview of running European research). The final chapter 5 is the result of a discussion among the Task group chairmen. It identifies the needs for further concerted research and breeding and gives recommendations for the chain-wide approach. This evolves into the expression of interest regarding the mycotoxin control problem in which several of the participants of the concerted action participate. The group has not drawn up a protocol for use throughout the chain, since organisations such as EUREPGAP and the Codex Alimentarius, have already studies underway for establishing such protocols. The data in this report may be used to underpin such protocols as needed.

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2. Crop production2.1 IntroductionMinimizing mycotoxin contamination starts with clean starting material, i.e. seed. The primary production is at the start of the chain and sits at the basis of any program to reduce the risk of mycotoxins further down the chain and to the end user, albeit human or animal. Breeding for resistance to Fusarium head blight (FHB) in wheat and Fusarium ear rot in maize is the potentially most rewarding strategy, but awkwardly difficult and timeconsuming. In this chapter various aspects of breeding for resistance are considered in relation to sustainability and mycotoxin accumulation. Fungicides have a long history in controlling FHB but with various degrees of success. Efficacy in relation to especially time of application is discussed. Resistance levels may vary between varieties depending on their location throughout Europe. To critically assess this supposed variation, a ring test with a set of common European wheat varieties was performed as part of the concerted action project. In this test attention is focussed on the relationship between (partial) resistance and mycotoxin accumulation.

2.2 Breeding for resistance to Fusarium spp. in wheat T. Miedaner1Fusarium head blight (FHB) is caused by several Fusarium species. Fusarium species are economically important pathogens in most agricultural crops. They occur on all vegetative and reproductive organs of plants causing wilts, rots or blights. Moreover, they have been isolated from soils of every continent except Antarctica (Windels, 1992). In small-grain cereals, about 20 Fusarium species have been regularly associated with disease symptoms (Duben & Fehrmann, 1979a; Gerlach & Nirenberg, 1982). F. culmorum (W.G. Smith) Sacc., F. graminearum Schwabe [teleomorph: Gibberella zeae (Schw.) Petch], and F. avenaceum (Corda ex Fries) Sacc. [teleomorph: Gibberella avenacea Cook] were most frequently isolated (Cook, 1968; Duben & Fehrmann, 1979a; Mesterhzy, 1995a). This introduction will focus on F. culmorum and F. graminearum because F. avenaceum isolates were generally found to be less aggressive in small-grain cereals (Colhoun et al., 1968; Mesterhzy, 1978; Duben & Fehrmann, 1979b; Diehl, 1984; Stack & McMullen, 1985; Wilcoxson et al., 1988).1 This version is specially updated by the author for this document and has been published before as part of the paper Breeding wheat and rye for resistance to Fusarium diseases in Plant-Breeding 116 (1997) 201-220.

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F. culmorum and F. graminearum are generalists, infecting all cereal species including wheat (Triticum aestivum L.) and rye (Secale cereale L.), and a large number of non-gramineous hosts (Gerlach & Nirenberg, 1982). F. culmorum was isolated frequently in the cooler maritime regions of Northern Europe (Parry et al., 1995), whereas F. graminearum is the predominating species on a global basis. The kind of disease they cause is predominantly a function of inoculum, plant growth stage, and environment (Cook, 1981a). Two ecologically and genetically different subpopulations of F. graminearum were described by Purss (1971) and Francis & Burgess (1977) and originally designated as Groups I and II. Recently, Group 1 isolates have been classified as the new species Fusarium pseudograminearum as judged by conidial morphology and molecular markers (Aoki & ODonnell, 1999). Seedling blight caused by F. culmorum and F. graminearum is mainly seed-borne in the humid climates and leads to a reduced number of plants and secondary attack by pests (e.g. Oscinella frit) due to thinned stands and delayed plant development. Seedling blight caused by F. pseudograminearum results, in contrast, from soil-borne infection and occurs in dry soils only (Burgess et al., 1981). At the stem base of small-grain cereals three epidemiological distinct Fusarium-incited diseases may occur: (1) brown foot rot caused by F. culmorum and F. graminearum in areas with high soil moisture and humidity (Cook, 1981a), (2) crown rot caused by F. pseudograminearum under dry weather and soil conditions, especially in the North-western U.S.A. (Cook, 1968), Eastern Australia (Burgess et al., 1981), South Africa (Marasas et al., 1988), and (3) common root rot caused by a complex attack of F. culmorum, F. graminearum and Bipolaris sorokiniana in the Great Plains of the U.S.A. and the Prairie Provinces of Canada (Windels & Holen, 1989). The attack of brown foot rot starts from aboveground inoculum (Cook, 1981a). The fungus penetrates the successive layers of the leaf sheaths during the growth period and finally reaches the stem. The lowest internodes, but not the crowns or roots, show necrotic lesions and may develop a soft rot that cannot be seen before flowering (Fehrmann, 1988). Brown foot rot is often the result of a complex attack of F. culmorum, F. avenaceum, Pseudocercosporella herpotrichoides and Microdochium nivale (Duben & Fehrmann, 1979a; Miedaner et al., 1993b). Brown foot rot causes yield losses due to a reduced capacity of the stem for the movement of water and nutrients and an increased risk of lodging. Additionally, lodged wheat and rye crops impair baking and feeding quality. Crown rot is caused by belowground inoculum entering the plants around emerging roots and crowns (Cook, 1981a). The infection remains latent unless the plant is subjected to heavy water stress reaching plant water potentials between -1.5 and -2.5 MPa (Cook, 1981b). The characteristic symptoms are scattered, bleached, and dead plants amongst unaffected plants on fields exposed to water stress. Crown rot causes premature ripening and thus a reduction of kernel number and/or kernel weight (Burgess et al., 1981). In the more humid climate of Central Europe the combination of heavy water stress and crown 8

rot is unlikely to occur in wheat (Jenkins et al., 1988), but in dry wheat growing areas crown rot is the most destructive stem disease (Cook, 1981a; Burgess et al., 1981; Wildermuth & McNamara, 1994). Fusarium species are usually not primary pathogens of healthy upper leaf blades but they frequently enter lesions caused by powdery mildew (Mathis et al., 1986) and aphids, or through mechanical wounds (Diehl, 1984). Head blight is caused by ascospore or macroconidia infecting in periods with high humidity (>92-94% relative humidity, Cook, 1981a) and temperatures above 15oC (Parry et al., 1995). Infections may occur at any time from head emergence to maturity, but disease severity is highest when inoculum is present in the flowering period of both wheat and rye (Anderson, 1948; Diehl, 1984; Mielke, 1988; Gang, 1996). Symptoms and disease development were extensively reviewed (Cook, 1981a; Teich, 1989; Sutton, 1982; Parry et al., 1995). Head blight epidemics may result in severe yield loss by destruction of the embryo and/or reduction of kernel weight, poor milling and baking quality of wheat (Meyer et al., 1986; Pomeranz et al., 1990), or reduced germination rate and seedling vigour in the following crop (Manka, 1989). Devastating epidemics occurring since the early 1990s in Midwestern USA resulted in yield losses up to 30%, a severe reduction in grain quality and high mycotoxin contents (McMullen et al., 1997; Windels, 2000). F. culmorum and F.graminearum are both capable of producing trichothecene type A toxins (HT-2 toxin, T-2 toxin), type B toxins (mainly deoxynivalenol, 3acetyldeoxynivalenol, 15-acetyldeoxynivalenol, nivalenol, fusarenone-X, calonectrin), and zearalenone in epidemics in wheat, barley, triticale, and rye (Marasas et al., 1984; Chelkowski, 1989; Perkowski et al., 1995). Several mycotoxins may occur simultaneously in different composition and amounts. They are hazardous to animal and human health (Friend & Trenholm, 1988; Pomeranz et al.; 1990, Snijders, 1990a; D'Mello et al., 1999). Yield losses caused by the various Fusarium diseases were reported to range for natural infections from 7 to 17% for seedling blight (Greaney et al., 1938; Duben, 1978), from 10 to 30% for foot rot (Duben, 1978; Meyer, 1985), from 0 to 17% for crown rot (Dodman & Wildermuth, 1987), and from 0 up to 30 - 70% for head blight (Martin & Johnston, 1982). With artificial inoculation much higher losses can be gained for the various diseases (e.g. Purss, 1966; Diehl, 1984; Miedaner & Walther, 1987; Chelkowski, 1989; Snijders, 1990b; Miedaner et al., 1993a). Fungicide treatment and agricultural management practises are only reducing the damage but cannot prevent yield and quality losses (Mielke, 1988; Teich, 1989; Milus & Parsons, 1994). Thus, the development of cultivars with appropriate disease resistance is the most effective means of controlling Fusarium diseases. The state of knowledge on F. graminearum and F. culmorum has been thoroughly reviewed with regard to symptomatology and epidemiology (Cook, 1981a; Burgess et al., 1981; Sutton, 1982; Jenkins et al., 1988; Teich, 1989; Miller, 1994; Parry et al., 1995), breeding strategies (Miedaner, 1997; Bai & Shaner, 1994; Mesterhazy et al., 1999), and toxicology (Marasas et al., 1984; Chelkowski, 1989; Pomeranz et al., 1990). 9

Mycotoxin production in wheat caused by Fusarium ssp. F. graminearum produces a variety of mycotoxins, namely nonmacrocylic trichothecenes and the estrogenic zearalenone (ZEA). Among these, deoxynivalenol (DON) and its derivatives 3-acetyl DON and 15-acetyl DON, ZEA and, in some parts of the world, also nivalenol (NIV) are most often encountered in wheat (Tanaka et al., 1988; Mirocha et al., 1989; Scott, 1990; Placinta et al., 1999). The co-occurrence of several of these mycotoxins in grain has often been reported (e.g. Mller & Schwadorf, 1993; Mirocha et al., 1994) and the frequency of mycotoxin-producing isolates in natural populations seems to be high. Of 114 isolates of F. graminearum collected from soil or cereals on a world-wide basis, 95 and 89% were capable of producing DON and ZEA in vitro, respectively (Mirocha et al., 1989). In smaller percentages, however, other patterns can be found. Some isolates of F. graminearum produce NIV, but they were found only rarely in the U.S.A. (Miller et al., 1991). They are more common in Japan (Ichinoe et al., 1983; Miller et al., 1991), but have also been detected in Hungary, Poland, and Italy (Miedaner et al., 2000). And although DON and NIV are chemically related, DON producers do not produce NIV and vice versa (Ichinoe et al., 1983; Miedaner et al., 2000a). ZEA can be found in small-grain cereals, the appearance of high amounts seems to be associated predominantly with corn (Yoshizawa, 1991). Out of 2403 samples of small-grain cereals analysed world-wide, about 20% were found to contain ZEA (Yoshizawa, 1991). F. culmorum, F. graminearum and F. pseudograminearum produce similar mycotoxins with minor differences in the relative amounts of each mycotoxin (Blaney & Dodman, 1988; Marasas et al., 1984; Snijders, 1990a; Atanassov et al., 1994). The ability of F. culmorum to produce NIV has only recently been detected (Atanassov et al., 1994; Mirocha et al., 1994). For both F. graminearum and F. culmorum, the mycotoxins produced and the amount of mycotoxin production highly depends on the isolate investigated (Marasas et al., 1984; Miller et al., 1991; Atanassov et al., 1994; Miedaner et al., 2000). It has been suggested that DON may contribute to the pathogenicity of F. graminearum and F. culmorum. DON inhibits protein synthesis (Miller, 1989) and growth of wheat coleoptile tissue and seedlings (Bruins et al., 1993). However, it appears that DON production may not be required for pathogenicity, i.e. the ability to cause disease, because F. graminearum isolates that were unable to produce DON and 3-acetyl DON in vitro were pathogenic on wheat, rye, and triticale seedlings (Manka et al., 1985). Moreover, a simple qualitative reaction (DON/no DON) cannot explain the quantitative nature of aggressiveness within F. graminearum populations as reported earlier (Miedaner & Schilling, 1996; Mesterhzy et al., 1999). According to Vanderplank (1984), aggressiveness designates the quantity of disease induced by a pathogenic isolate on a susceptible host when the isolates do not 10

interact differentially with host cultivars. Thus, DON might have no relation to pathogenicity, but may contribute to aggressiveness, i.e. the extent of fungal colonisation within host tissue in the early stages of pathogenesis (Snijders & Krechting, 1992). This was recently demonstrated by the generation of a trichothecene-deficient isolate of F. graminearum that was less aggressive than the DON-producing wild type (Proctor et al., 1995; Desjardins et al., 1996). Interestingly, the trichothecene-deficient isolate was still able to produce symptoms, i.e. its pathogenicity was retained (Proctor et al., 1995). The physiological factors leading to different aggressiveness levels among isolates are still unclear. Besides mycotoxins, cell-wall-degrading enzymes may also play a major role.

Susceptibility of wheat varieties to head blight the problem in wheat products at the beginning of the production chain Fusarium culmorum and F. graminearum are the main causal organisms of head blight in wheat (Triticum aestivum L.), rye (Secale cereale L.), and triticale (x Triticosecale Wittmack) in humidtemperate climates (Snijders, 1990a). Complete resistance appears to be rare but large quantitative variation for head blight resistance in winter wheat was found for both F. culmorum and F. graminearum in all varieties tested. Hanson et al. (1950) summarised the results of former U.S. evaluation trials across thousands of entries and reported that all genotypes became infected, i.e. no source of complete resistance was found. Moreover, most genotypes proved to be susceptible with only few exceptions. Similar conclusions were drawn from more recent tests (Walther, 1976; Miedaner & Walther, 1987; Mesterhzy, 1987; Mielke, 1988; Tomasovic, 1989; Snijders, 1990b; Saur, 1991; Bai & Shaner, 1994; Mesterhzy et al., 1989). Durum wheat varieties were generally more susceptible than bread wheat but some resistance was recently reported(Stack et al., 2001). Distinct resistance sources for F. graminearum-incited head blight in hexaploid wheat were reported from three origins: winter wheats from Eastern Europe, spring wheats from Japan and China (e.g. 'Nobeokabozu Komugi', 'Sumai 3', 'Ning' selections), and from Brazil (e.g. 'Frontana', 'Encruzilhada') (Schroeder & Christensen, 1963; Mesterhzy, 1987). Snijders (1990b) confirmed resistance of some of these wheat materials for infections to F. culmorum and identified additional accessions from these gene pools. Despite a high genotypic variance, genotype-by-environment interaction plays a major role in the wheat/Fusarium head blight pathosystem (Mesterhzy, 1987, 1989, 1995a,b; Miedaner et al., 2001b). Therefore, correlations of host resistance to F. graminearum between years may vary considerably. In experiments over six years, Mesterhzy (1995a) reported correlation coefficients between each of two years ranging from 0.19 to 0.81 for head blight rating and from 0.32 to 0.67 for relative grain weight. Stability of resistance expression over environments greatly depended on the resistance level of the genotypes studied. Highly resistant materials showed less variation across environments than medium susceptible genotypes (Mesterhzy1995a). These data indicate that tests across several 11

environments are necessary to rank genotypes properly for their resistance to Fusarium head blight. In quantitative-genetic experiments, a preponderance of additive variance for resistance to F. graminearum and F. culmorum have been found (Gu, 1983; Bai et al., 1993; Ban & Suenaga, 2000; Snijders, 1990c,d, respectively). In addition the mean head blight resistance of F2 populations can be predicted by the resistance of the parental lines (Snijders, 1990d,e). The only exceptions were found for progenies from crosses where one awned genotype was involved (Snijders, 1990d). Within the non-additive components of genetic variation, dominance was found most often. However, dominance expressed as heterosis for resistance was significant in some F1 crosses only (Hanson et al., 1950; Tomasovic, 1989; Snijders, 1990c). Similarly, the occurrence of epistatic effects of the additive-by-additive component was reported for a minority of crosses (Snijders, 1990d; Bai et al., 1993). Nakagawa (1955) firstly published an estimate of the number of effective factors by generation mean analysis. He found three genes that controlled scab resistance. In more recent surveys, Bai & Xhiao (1989) and Bai et al. (1993) reported one to three genes responsible for resistance to F. graminearum head blight in Chinese materials. Snijders (1990d) found on the basis of 45 crosses that the number of effective factors varied from one to six for F. culmorum head blight. It should be noted that, from the theoretical point of view, all these estimates are only a rough indication of the true number of genes responsible for Fusarium head blight resistance. At least three assumptions cannot be fulfilled in the experiments: (1) equal gene action, (2) one parent supplies only positive, the other only negative alleles, and (3) equal degree of dominance (Wright, 1968). Moreover, in any one cross only a limited sample of genes contributes to segregation and, therefore, the real number of genes will most likely be underestimated (Geiger & Heun, 1989). High genotype-by-environment interaction will additionally affect the estimation of gene number when the experiments are not conducted in several environments. Molecular marker analyses of some resistance sources revealed one to five quantitative-trait loci (QTL) for head blight resistance up to now (Kolb et al., 2001). Bai et al. (1999) analyzed 133 RILs derived from a cross of `Ning7840' (resistant) x `Clark' (susceptible) with AFLP markers and identified one major QTL for scab resistance that explained almost 60% of the phenotypic variation for Type II resistance in that population. Later, this QTL was localized on chromosome 3BS. Waldron et al. (1999) identified several QTL for scab resistance by analyzing RFLP markers in 112 F5-derived RILs from the cross between `Sumai 3' (resistant) and `Stoa' (moderately susceptible). Two major QTL were located an 3BS of `Sumai 3' and 2AL of `Stoa', respectively. The most infonnative RFLP marker in the 3BS rcgion explained 15% of the plienotypic variation in that mapping population (Waldron et al., 1999). Later, Anderson et al. (2001) reported several microsatellite markers linked to the same QTL for scab resistance an 3BS. Subsequently, other groups have confirmed the association of markers with a QTL on 3BS (Buerstmayr et al., 2002; Chen et al., 2000; Gupta et al., 2000, 2001; Zhou et al., 2000). Buerstmayr et al. (2002) reported three chromosome regions associated with FHB resistance in a double12

haploid mapping population with `Sumai 3' as resistant parent. Again, the most-prominent effect was detected an chromosome 3BS, explaining up to 60% of the phenotypic variation for type II resistance (Buerstmayr et al., 2002). When analysing type I and II resistance together by spraying a conidia suspension onto the heads, two QTLs on 3B and 5A each were found to explain about 30% of phenotypic variance (Buerstmayr, pers. comm.).

Resistance to mycotoxin accumulation as a future breeding target in wheat breeding Host genotypes suffering Fusarium head blight might accumulate several mycotoxins in their grains (see Mycotoxin Production earlier). Most commonly, DON and its metabolites were found on a worldwide basis (Snijders, 1990a). In naturally infected grain, mean DON concentrations of wheat samples collected arbitrarily ranged from 0.03 to 1.78 mg kg-1 with maximum values between 0.14 to 8.53 mg kg-1 (Snijders, 1990a). In a five-year analysis of wheat in Southwest Germany, Mller & Schwadorf (1993) found a mean DON content of 1.6 mg kg-1, ranging from 0.004 to 20.5 mg kg-1. In artificial Fusarium inoculations much higher DON concentrations have been reported in wheat (e.g. Mesterhzy & Bartok, 1993; Trissler, 1993). In a collaborative analysis of six wheat, six triticale and 12 rye genotypes, rye and triticale accumulated a comparable amount of DON, but wheat showed three times higher DON contents across two locations although disease severity in wheat was somewhat lower than in rye (Miedaner et al., 2001b). Because DON was detected also in healthy looking wheat (0.2-5.9 mg kg-1) and rye (0.10.8 mg kg-1) kernels (Perkowski et al., 1990, 1995, respectively), and keeping in mind that mycotoxin analyses are expensive, an association between resistance traits and mycotoxin accumulation would greatly enhance progress in selection of less toxin-accumulating genotypes. Most studies found a low to medium correlation between resistance traits (such as head blight rating, relative grain weight or thousand-grain weight) and DON content in inoculation experiments. However, disease incidence (% diseased heads per plot) and the seed infection rate seem to result in somewhat higher correlations to DON content than other factors. In most studies, the environment (location-year combination) strongly influenced the correlation. Environmentally stable conditions were only found in one winter wheat cross of largely differing parents (Miedaner, unpubl.) and in the study of Mesterhzy & Bartok (1993). A biometrical cause for only moderate correlation between resistance traits and DON accumulation might be the considerably higher genotype-environment and error variances of DON content (Miedaner et al., 2001b) leading to a large bias in correlation estimates. Other causes might be rooted in the disease epidemiology. In years when disease is severe, 13

or in highly susceptible genotypes, kernel number per head will be low due to loss in threshing of severely shrivelled grains or their early abortion in the head. In such cases, mycotoxin content of grain samples may be underestimated. Additionally, when infections occur early, F. culmorum can invade the primary bundles in the head. The head sections above the infection site turn white due to water and nutrient depletion without being colonised (wilting). Thus, mycotoxin contamination is unlikely to occur in those parts of the head. Moreover, symptom expression or yield reduction and mycelial growth and mycotoxin production might be influenced, at least partially, by different environmental conditions. This hypothesis is supported by the moderate correlation between head blight rating and ergosterol content of infected heads (Trissler, 1993; Miedaner & Perkowski, 1996). Moreover, the correlation between a resistance trait and mycotoxin content depends on the fungal isolate used as reported for three and six individual Fusarium spp. isolates, respectively (Snijders & Perkowski, 1990; Atanassov et al., 1994). In general, highly resistant wheat genotypes such as Sumai 3, Wuhan 37E-OY-OFC, Wuhan 10B-OY-OFC from China, Nobeokabozu from Japan and materials developed from these sources showed very low DON contents even under favourable epidemic conditions (Mirocha et al., 1994; Mesterhzy, 1995b; Mesterhzy et al., 1999). However, the level of resistance in most breeders' materials is lower. In particular, moderate susceptible genotypes may sho high deviations from the mean regression of resistance traits on DON content (Teich et al., 1987; Mesterhzy et al., 1994).

Durability of Fusarium resistance of wheat varieties The success of resistant varieties in practise depends on the durability of the improved resistance. However, experimental results on this aspect are very limited for the wheat, rye/Fusarium pathosystems. Durability of resistance depends on pathogen variation, mechanisms and inheritance of resistance, and agricultural management practises (Parlevliet, 1993; Bowden & Leslie, 1994 ). However, the occurrence of specific adaptation of certain F. culmorum or F. graminearum isolates to a host cultivar seems to be unlikely because (1) a low degree of pathogenic specialisation was reported, (2) both Fusarium species are good saprophytes in soil habitats, (3) no consistent isolate-by-host genotype specificity was found in wheat and rye, and (4) host resistance was shown to be inherited quantitatively with no single genotype being completely resistant. Selection pressure on a Fusarium population, therefore, should be small. Even if a high level of quantitative resistance might cause a change in the composition of Fusarium populations in future, erosion of resistance should most likely be stepwise and slow making adequate resistance available for acceptable periods of time. These theoretical considerations are supported by experimental results of Mesterhzy (1995a) who reported head blight resistance to be stable for one highly resistant genotype when tested across sixteen environments. However, the ultimate test of durability of resistance is to grow the resistant variety over a

14

longer period of time on a great acreage in areas where the disease occurs regularly (Johnson, 1993).

Conclusions from breeding for resistance to Fusarium spp. in wheat Taking all experimental data together, resistance breeding to Fusarium diseases is not limited by the lack of genetic variability but by the limited selection response. Thus, mapping resistance gene complexes by DNA markers should provide a solution to this problem. Analysis of quantitative trait loci (QTL) are used by several groups to determine the number of genes involved, to assess gene action and interaction, to investigate the correlation between resistance and other agronomic traits, and finally to study the interactions with plant organs, plant growth stages and environments at the individual QTL level (see above). Molecular markers could be further valuable in transferring important QTLs from exotic germplasm to adapted breeding materials (marker-assisted backcrossing), or selecting within progenies of crosses between susceptible and resistant genotypes (marker-assisted selection). However, the precision of mapping QTLs for Fusarium resistances greatly depends on the heritability of resistance assessment, the number of effective factors, the distribution of QTLs across the genome (linkage) and the occurrence of non-genetic factors (Van Ooijen, 1992). In particular the high importance of host genotype-by-environment interaction and the association of host resistance with plant growth stage require a high experimental input (large number of environments, different inoculation treatments) for properly estimating the genotypic values needed for a precise QTL mapping of Fusarium resistances. Doubled-haploid (DH) techniques might offer a further approach to enhance selection efficiency. DH lines derived from F1 crosses are completely homozygous. They allow selection for Fusarium resistance in multi-environmental tests with a maximal genetic variance between homogeneous entries and, therefore, a precise estimation of the genotypic value. Because this is not possible for selection in segregating generations, DH techniques would offer a perfect solution to select for quantitative resistant genotypes. A fast recurrent selection (RS) scheme could be realized that would be especially advantageous for inbreeding crops (Foroughi-Wehr & Wenzel, 1990). Genetically, a RS scheme based on the DH technique would be most advantageous when the resistance is mainly governed by recessive genes and these are not closely linked to undesired agronomic traits, because the probability of recombination between closely linked genes is lower in DH steps than by subsequent selfing (Becker, 1993). The inheritance studies showed a low importance of dominance for most of the pathosystems reported. A linkage to agronomically undesired traits is most probably occurring when the resistance genes are being introgressed from exotic germplasm. Then, the occurrence of undesired linkages should be either experimentally tested or the first cycle(s) of RS should be done by singleseed descent. A maximal selection gain would be achievable, if DH techniques could be 15

combined with efficient marker-assisted selection. A reliable selection for Fusarium resistances would then be possible directly with DNA from the regenerated single plants. Although the DH technique can be used successfully in wheat, this is not yet feasible for winter rye caused by the low regeneration rate in adapted breeding materials (FlehinghausRoux et al., 1995). Progress in improving Fusarium resistance may be gained in future by gene technology. Several procedures are under development, e.g. the transfer of defense-response genes, including those for anti-fungal proteins, and of genes for detoxification of DON and ZEA, the enhancement of the efficiency of transporter proteins within the plant cell for a rapid export of mycotoxins, and the production of an artificial avirulence by manipulating the host-pathogen recognition (for review see Dahleen et al., 2001). Another subject of interest for the breeder is whether reduced susceptibility of the host genotypes to Fusarium head blight will necessarily result in a correlated reduction of the mycotoxin content in the grains. This depends on the correlation between resistance traits and mycotoxin contents. Moreover, the number and relative importance of different mycotoxins should also be considered in future studies. Although DON is reported to be the most prevalent mycotoxin in F. culmorum and F. graminearum infections of small-grain cereals, seven out of 42 tested F. culmorum isolates were capable of producing high levels of nivalenol on a susceptible winter rye genotype (Gang, 1996). In addition, F. graminearum isolates can secrete high amounts of zearalenone (Marasas et al., 1984). Considering the high importance of mycotoxin contamination for animal and human health, the interactions between Fusarium isolates, host genotypes, mycotoxin accumulation and environment should be analyzed in more detail. The most serious lack of knowledge in resistance genetics of Fusarium diseases concerns the causes of host resistance and pathogen aggressiveness. For all Fusarium diseases in small-grain cereals, less susceptible host genotypes can be identified, but little is known about the molecular or physiological basis of the resistance mechanisms. The relative contribution of preinfectional mechanical barriers or postinfectional host defenses is also unknown. Similarly, the role of the mycotoxins in pathogenesis is still not clear. Does a highly aggressive isolate cause more disease because it produces more toxin, or does it produce more toxin because it causes more disease (Yoder, 1981)? This could only be answered if the kinetics of mycotoxin production during the very early processes of pathogenesis are monitored by highly sensitive assays. Additionally, the existence and possible role of other factors that may be responsible for aggressiveness, such as cell-wall degrading enzymes, hormones, or specific metabolites altering the host's resistance reaction, are not known. All these questions do not substantially impede selection efficiency for Fusarium resistance but their answer would greatly contribute to our understanding of the fascinating cereal-Fusarium interaction and may offer new approaches for resistance breeding.

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2.3 Ring test with selected European winter wheat varieties P. Ruckenbauer, T.W. Hollins, H.C. de Jong and O.E. ScholtenGenetic variation in resistance to the disease is well recognised in most parts of the world (Bai et al., 2001; Miedaner et al., 1999; Snijders, 1990b). Variety registration procedures in a number of countries assess genetic resistance to fusarium head blight and minimum standards for resistance are in place (Bundessortenamt, Beschreibende Sortenliste, 2000, Hannover; NIAB, UK Recommended Lists of Cereals, 2000). For example such differences in resistance can be demonstrated clearly between 37 commercially grown varieties at the Monsanto Cambridge site in the trial year 2000 (Table 1). Wheat varieties are most susceptible at the flowering stage, growth stage (GS) 61 69, (Tottman & Broad, 1987), so it is important when critically comparing varieties to inoculate at a consistent stage of growth. In this way lines with different maturity, often from different geographic regions, can be compared. In this experiment disease was then assessed at defined intervals after inoculation (350, 400 & 450 o centigrade days), which accounted for average temperature as well as time to minimise inaccuracies due to changing average temperatures during disease development. Large differences in resistance among current European varieties were observed. In these very disease conducive conditions up to 60% infection was observed on some UK varieties (Charger) whereas the widely grown German variety Batis had 20% disease only. Several lines were even less diseased (Sumai#3, etc.) but most of these are low yielding and/or poorly adapted to northwest Europe. Their resistance is currently being incorporated in breeding programmes throughout the world. Stability of resistance Among the wheat breeders of the participants of Mycotochain (Monsanto at Cambridge, UK, IFA-Tulln at Tulln, Austria and Cebeco Seeds at Lelystad, The Netherlands) a ring test with currently grown winter wheat varieties within the EU was performed. These field trials were sown at locations in Cambridge, UK; Tulln, Austria; Lelystad, The Netherlands and Wallerfing, Germany, with variable treatments concerning irrigation and inoculation. Within the ring test trials seventeen varieties from five different European countries were tested at four locations, some with artificial infection and some with natural infection. Disease estimates demonstrated that, in general, varieties ranked similarly at the different 17

sites (Table 2) although some varieties (e.g. Semper) showed some inconsistency across sites. Results of Wallerfing are not presented because infection levels were too low. Table 1. Amount of Fusarium head blight on winter wheat varieties after artificial inoculation with Fusarium culmorum in Cambridge in 2000. Varieties inoculated individually at anthesis and assessed for disease by thermal time.Variety 95NYP1256 Sumai#3.2 Zhefeng Ning 7840 Patton Heyne Jagger Freedom Cockpit Piko Soissons Ludwig Kraka AC Winsloe Greif Frelon Petrus Achat Astron Batis Isengrain Huntsman Tremie Smart Savannah TP1689/-/-/18 Fruhgold Semper Ritmo Rialto Contra Shango Consort Tower Equinox Hanseat Charger 5% LSD Date inoculated1 3502 143 141 141 148 148 152 143 150 160 164 152 160 164 154 160 154 160 164 164 164 154 160 152 164 164 164 157 164 164 160 160 164 164 164 160 160 160 0.2 0.2 0.5 1.8 0.7 0.6 0.3 3.9 2.7 1.6 3.4 3.9 3.6 4.0 7.3 6.0 3.0 2.8 6.1 5.0 10.9 10.9 12.7 7.3 8.7 13.1 12.1 8.8 18.6 15.5 15.6 19.7 15.3 13.1 26.4 30.8 30.6 Assessed at: (C days) 400 0.3 0.3 0.6 1.8 2.5 3.2 5.7 5.8 7.4 9.9 8.0 12.0 11.9 8.6 10.5 9.2 19.1 17.3 15.7 21.5 17.3 26.4 22.7 27.9 28.8 31.5 29.9 34.3 32.7 36.6 38.1 39.6 44.7 46.7 55.1 54.6 65.8 12.5 (within date) Average disease3 450 1.5 1.7 1.7 3.2 6.4 9.2 9.9 12.3 13.7 20.3 21.9 20.0 21.8 24.7 20.2 23.1 24.3 27.8 26.8 33.3 35.6 35.5 40.9 47.1 50.5 45.5 49.6 49.6 43.2 58.0 58.8 61.2 69.5 70.0 68.8 73.6 80.4 1 1 1 2 3 4 5 7 8 11 11 12 12 12 13 13 15 16 16 20 21 24 25 27 29 30 31 31 31 37 37 40 43 43 50 53 59 7

Inoculated with a mixture of two F. culmorum isolates in 6 replicates and grown under irrigation

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1 2 3

days after 1 January average of daily maximum and minimum temperature summed over days percentage of ear area with symptoms of FHB

Table 2. Percentage of disease on heads of winter wheat at four locations in Europe carried out in 2000 under artificial infection in the UK and Austria and natural infection in Germany and The Netherlands. Variety Cambridge, UK F.c1. Soissons Ludwig Petrus Achat Batis Isengrain Tremie Ritmo Semper Contra Shango Consort Tower Hanseat Charger Equinox Bercy 5%LSD1

Tulln, Austria F.c1. 22 19 14 26 18 26 58 54 29 46 54 32 21 76 76 62 83 12.3 F.g.2 21 24 15 32 15 26 58 58 42 42 62 46 20 62 76 46 83 14.7

Lelystad, Netherlands F.c1. 7 4 1 4 3 6 17 13 4 11 14 9 3 23 28 11 30

11 12 15 16 20 21 25 31 31 37 40 43 43 53 59 50 8.8

Fusarium culmorum; 2 Fusarium graminearum

In addition in Austria varieties were tested against six separate pathogens that have the potential to cause FHB (F. culmorum, F. graminearum, F. avenaceum, F. poae, F. sporotrichoides, F. subglutinans). Only two species caused substantial disease on the susceptible lines (see Table 2) and again variety ranking was similar. These results are in agreement with those reported by Van Eeuwijk et al. (1995) who similarly tested varieties covering a range of resistance to different pathogens in different regions of Europe.

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Relationship between disease and mycotoxin content Specific end use requires grain free from mycotoxins so it is important to the grower, and plant breeder, to know that decreased disease in the field leads to decreased mycotoxin content in the grain. Several studies have show this to be the case with either natural epidemics (Wosnitza, pers. comm.) or with artificial infection (Bai et al., 2001; Miedaner et al.,, 1999), the latter often when disease levels, and mycotoxin content, are high. To confirm these observations, the same three partners of Mycotochain analysed the DON content of the 17 wheat samples of the ring trial by means of GC and HPLC methods (Table 3). In situations where M. nivale is involved, a high visual infestation in the field does not always mean that mycotoxins are present. It was noted that the variety Petrus, with the best field scores (Table 2), showed the lowest DON content across all trial sites whereas the samples of the most susceptible winter wheat variety of the ring test Hanseat had the highest DON contents, independently from sites and treatments. Table 3. DON content of wheat samples in ppm and in % of the experimental mean determined in samples taken from 4 locations in 2000. Cultivar Petrus Soissons Tower Batis Achat Isengrain Ludwig Semper Contra Bercy Tremie Equinox Consort Charger Ritmo Shango Hanseat LocationMean Tulln (A) ppm 2.51 6.05 4.68 3.44 10.59 5.29 17.94 15.85 15.97 33.42 24.03 16.05 31.91 27.42 27.07 26.79 36.00 % 14 34 26 19 59 29 100 88 89 186 134 89 178 153 151 149 201 Cambridge (UK) Lelystad (NL) Wallerfing (D) ppm 13.02 10.95 13.45 21.88 18.30 32.68 26.21 29.23 27.20 21.54 36.00 36.00 32.55 36.00 32.67 36.00 36.00 27.04 % 48 40 50 81 68 121 97 108 101 80 133 133 120 133 121 133 133 100 ppm 2.32 3.03 4.92 5.72 7.09 3.05 5.15 7.28 15.00 13.09 11.91 23.54 16.70 18.62 25.89 24.39 30.06 12.81 % 18 24 38 45 55 24 40 57 117 102 93 184 130 145 202 190 235 100 ppm 0.20 0.60 1.09 0.36 1.06 0.85 1.16 0.64 0.56 0.77 1.10 0.97 1.72 0.97 1.02 2.08 0.74 % 21 64 117 38 113 91 124 69 60 82 117 104 184 104 109 222 79 Mean over Locations ppm 4.51 5.16 6.03 7.85 9.26 10.47 12.61 13.25 14.68 17.20 18.26 19.14 20.72 20.75 21.66 22.31 25.70 14.68 % 31 35 41 53 63 71 86 90 100 117 124 130 141 141 148 152 175 100

17.94 100

0.93 100

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The correlation between DON and percentage of diseased spikelets after artificial inoculation in Tulln is 0.80 (Figure 1). This result clearly indicates that the levels of DON in resistant varieties in general are lower than in susceptible varieties. The Tulln samples were furthermore subjected to two DON analysis methods: GC (IFA-Tulln) and HPLC (PRI-Wageningen) in order to compare both methods (Figure 2). A high correlation of 0.87 was obtained between both analythical methods.40

30

DON (ppm)

20

10 R2 = 0.80 0 0 10 20 30 40 50 60 70 80 90

% diseased spikelets per plot (F. gram. )

Figure 1. Correlation between DON (ppm) and percentage of diseased spikelets after artificial inoculation with F. graminearum (Tulln, 2000).

21

40

DON in ppm measured by GC

30

20

10 R2 = 0.87 0 0 10 20 30 40 50

DON in ppm measured by HPLC

Figure 2. Correlation between DON (ppm) measured by HPLC and GC of diseased spikelets after artificial inoculation with F. graminearum (Tulln, 2000).

2.4 Control of the fungus through the use of fungicides P. JenningsControl of FHB by fungicides has been inconsistent. In practice fungicides are applied with the intention of controlling other head diseases as well as Fusarium and even when correctly timed for FHB may only be 60-70% effective. Agrochemical companies are addressing the problem of fungicide activity to FHB and improved active ingredients are continually being tested. There are several areas where important decisions have to be made in order to achieve optimal control of FHB.

Product choice FHB is associated with a complex of species on the ear, a product which shows good activity against one FHB pathogen may not be active against another so correct product choice is of particular importance. Such differential control of FHB pathogens was shown in experimental field trials carried out by Jennings et al. (2000). Trials inoculated at mid22

anthesis with a mixed conidial suspension of FHB pathogens showed that tebuconazole (as Folicur) effectively controlled the toxigenic Fusarium species present on the ear, but showed little control of the non-toxigenic M. nivale. However, the application of a strobilurin fungicide, azoxystrobin (as Amistar), controlled M. nivale but not the Fusarium species present. Similar differential control by tebuconazole and azoxystrobin has also been reported in trials naturally infected by FHB pathogens (Simpson et al., 2001). Differential control of fusaria and M. nivale also exists within the MBC group of fungicides, however this has arisen through the widespread development of resistance in populations of M. nivale (Locke et al., 1987; Pettitt et al., 1993). Other products with good efficacy towards fusaria responsible for FHB, include metconazole [as Caramba (Jennings et al., 2000)], prochloraz [as Sportak (Matthies & Buchenauer, 2000)], and epoxiconazole and carbendazim [as Opus and Derosal WDG respectively (Nicholson et al., unpublished data from HGCA project No. 2067). Many of the strobilurin fungicides, such as azoxystrobin, trifloxystrobin and kresoxim methyl, show good efficacy towards M. nivale. In most cases the reduction in disease symptoms which followed fungicide application also gave a reduction in deoxynivalenol (DON) contamination of grain compared to control plots. However, under certain circumstances it has been reported that application of some fungicides can lead to increases in DON levels in the field (Jennings et al., 2000). Trials carried out in 1998 and 1999 showed that following the application of azoxystrobin DON levels in grain increased in 1998, but not in 1999. The difference in the two years was the FHB pathogen mixture found on the ear. In 1998, M. nivale and F. culmorum were both detected on the ear in control plots, whereas in 1999 M. nivale was not detected. This suggests that the combination of FHB pathogens found on the ear is important in determining whether increased DON production occurs following the application of azoxystrobin. Where both M. nivale and F. culmorum were present on the ear the removal of M. nivale, through use of azoxystrobin, reduced competition on F. culmorum, resulting in increased DON levels in grain. Where M. nivale was absent from the ear, the application of azoxystrobin did not alter competition between FHB pathogens and had therefore no effect DON levels. Similar increases in DON have also been seen from 'on-farm' data (Turner et al., unpublished data). In 1998, a survey carried out on UK wheat grain indicated the predominant pathogen was M. nivale, with low levels of Fusaria also present (Turner et al., 1999). Examination of the fungicides applied to each field analysed for toxin and FHB pathogen indicated that where azoxystrobin was applied to the ear the level of DON was higher than where there was no azoxystrobin application.

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Timing of application Arguably the timing of fungicide application is more important than product choice when trying to control FHB and mycotoxin contamination of grain; no matter how effective the fungicide if it is applied at the wrong time it will not control FHB. Mid-anthesis is the most susceptible time for infection of wheat by FHB pathogens (Sutton, 1982) and as such is the most appropriate time to apply a fungicide spray aimed at FHB. Work carried out by Homdork et al. (2000) and Matthies & Buchenauer (2000) have both highlighted just how narrow the window for fungicide application is for optimum control of FHB pathogens. Matthies & Buchenauer (2000) investigated timing of fungicide application on disease development using trials artificially inoculated with F. culmorum at mid-anthesis. Fungicide treatments applied were tebuconazole or prochloraz at 8 days pre, 2 or 9 days post inoculation. The most effective treatment timing for both fungicides was 2 days post inoculation. The efficacy of the fungicide treatments decreased with increasing time interval between fungicide application and inoculation. A similar set of experiments carried out by Homdork et al. (2000) using F. culmorum inoculation with tebuconazole sprayed at either 3 days pre and/or 5 days post inoculation, again showed the closer the timing of the spray and inoculation, the better the efficacy of the fungicide. Some reports of fungicide failure in the field can be directly attributed to incorrect timing of application. Milus & Parsons (1994) concluded after testing the efficacy of seven fungicides against F. graminearum that the prospects for chemical control of head blight were poor. Fungicides were applied to plots at the end of heading growth stage. To each fungicide treated plot F. graminearum was inoculated three times, at the beginning, mid and end of anthesis (this equated to 2, 5 and 7 days post fungicide application). The results showed no reduction in levels of either head blight or DON following fungicide treatment. However, as already highlighted, inoculum landing on the ear seven days post fungicide treatment would not be effectively controlled. A more appropriate growth stage for the fungicide application would have been mid-anthesis. At this growth stage the time interval between fungicide application and inoculum arrival at the ear would be at its optimum at between two and three days.

Application rate To achieve the optimum efficacy against FHB pathogens a fungicide must be applied at the manufacturers recommended rate. Work carried out by Nicholson et al. (unpublished data from HGCA project No. 2067) showed that halving the rate of several fungicides led to significant reductions in control of FHB disease levels and mycotoxin production.

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At present, the challenge of controlling FHB and mycotoxin contamination of wheat grain will only be met through an integrated approach to crop protection. The use of fungicides forms an essential part of this approach. However, the inappropriate use of fungicides through incorrect product choice, timing of application or rate of product can significantly reduce the efficacy of a fungicide application.

Conclusions Differential control of FHB pathogens exists between fungicides. It is important to

determine which FHB pathogens are likely to be present on the ear in order to make an informed choice on the appropriate fungicide to use. In some instances it may be appropriate to apply a mixture of products. Timing of application is critical. For optimum control fungicides should be applied at mid-flowering. Fungicides should always be applied at the manufactures recommended rate. Any reduction in the rate applied will reduce the efficacy of the fungicide.

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2.5 Risk factors in Fusarium head blight epidemics E.T.M. Meekes and J. KhlIntroduction Fusarium head blight (FHB), also known as Fusarium ear blight or scab, can be linked with up to 17 causal agents in small grain cereals, although most records concern five species: Fusarium culmorum, F. graminearum, F. avenaceum, F. poae en Microdochium nivale (Parry et al., 1995). FHB is considered to be part of a complex of cereal diseases, since many of the species responsible for FHB can also cause seedling blight and foot rot. However, the epidemiological relationship between the three diseases is not always clear. Central to the disease cycle is the survival of these pathogens. The above-mentioned species, except for F. poae, are able to survive saprophytically on crop residues. The spores produced on this debris are considered to be the major source of FHB (Parry et al., 1994). Damaged caused by FHB can vary between regions, years and cropping systems. A combination of warm weather and rain before and during anthesis will enhance chances on FHB. In addition, several cultivation factors can influence the severity of a FHB epidemic like: 1) crop rotation, 2) soil preparation, 3) choice of wheat cultivar, 4) use of fungicides and/or plant grow promoters, 5) use of fertilizer, and 6) weed control (Bauer, 2000; Meier et al., 2000), not necessarily in this order. All Fusarium species are able to produce one or more mycotoxins, but M. nivale is not (Chelkowski, 1998). The irregular pattern of FHB epidemics has led to an underestimation of the potential danger of a toxin contaminated wheat crop (Snijders, 1990a).

Crop rotation In Europe as well as in North America, F. graminearum seems to replace other pathogens like F. culmorum and F. avenaceum as major causal agent of FHB (Clear & Patrick, 2000; Rintelen, 2000). This phenomenon is partly attributed to the inclusion of maize in the cropping system and an increase of short rotation intervals or abandonment of crop rotation al together (McMullen et al., 1997; Rintelen, 2000). Maize as previous crop of wheat provides a higher risk for FHB incidence and DON contamination of wheat than potato, sugarbeet, wheat, barley or soybean (Beck & Lepschy, 2000; Dill-Macky & Jones, 2000). The amount of decomposition of crop residues correlates negatively with the amount of Fusarium spores produced in spring especially when grain-maize is grown (Beck & Lepschy, 2000; Pereyra et al., 1999). Maize is not only a 26

good host for F. graminearum, its residues also take longer to decompose than for instance wheat residues and therefore providing ample opportunity for F. graminearum to survive the winter saprophytically (Beck & Lepschy, 2000). In Northwestern Europe the increase of silage maize acreage coincided with the increase of FHB caused by F. graminearum. In Bavaria (Germany), for instance this increase was 2-3 fold from 1972 to 1988 (Eder, 2000), in the Netherlands this increase was even more extreme 50 times increase in area from in 1970 to 1999 (Anonymous, 1970; Anonymous, 1999). In the 1990s cultivation of grainmaize also increased, posing a higher risk, because it leaves more residues in the field than silage maize (Beck & Lepschy, 2000). In North America an additional explanation for further increasing of FHB is the high percentages of cultivated acres planted to susceptible host crops and short rotations (McMullen et al., 1997).

Soil preparation Crop residues play a role in the disease cycle, especially when decomposition is slow. Residues at the soil surface decompose considerable slower than buried residues, increasing the inoculum potential of fungi causing FHB (Pereyra et al., 1999). The introduction of no-tillage practices to reduce soil erosion has led to an increasing amount of crop residue left at the soil surface, posing a higher risk for FHB. Moldboard plow treatments led to considerable lower crop residue cover, FHB incidence and DON content compared to chisel plow or no-tillage treatments (Dill-Macky & Jones, 2000). No tillage after a previous maize crop is the highest risk factor leading to high levels of DON contamination (Bauer, 2000; Dill-Macky & Jones, 2000).

Host range Besides crop residues there are several other possible inoculum sources of which their role is not exactly known. Other grasses, like ryegrass, timothy and fescue are susceptible to the same causal agents of head blight (e.g. (Engels & Kramer, 1996; Holmes, 1983). In most cases the fungi are associated with seedling diseases and foot rot, but some report also ear infection (Cagas et al., 1998). Grasses infected with Fusarium spp. also tested positive on toxin presence (Engels & Kramer, 1996). There is strong evidence that species causing seedling and foot diseases in grasses also cause the same diseases in wheat, but it is unknown what role they play in FHB. The same fungal species causing head blight have been isolated from several dicotyl weeds, all of these isolates were able to infect wheat. Weed control did lead to lower Fusarium infection, although this effect was not quantified (Jenkinson & Parry, 1994a). Fusarium spp. potentially causing FHB can also be isolated from green manure plants like clover and lupin. However, the role of other host plants of Fusarium spp. and M. nivale in FHB epidemics is unknown. 27

Weather conditions Crop rotation, soil preparation and presence of alternative hosts determine, among others, the inoculum potential at the beginning of the growing season and during anthesis. Severity of FHB is related to inoculum potential, but occurrence of FHB is highly dependent on weather conditions. If weather conditions for infection during anthesis are conducive the damage by FHB will be limited, despite high level of inoculum. Weather conditions influence different parts of the infection cycle, having the following influences on the complex of diseases caused by Fusarium spp. (Parry et al., 1995): Warm dry soil conditions during the early part of the growing season promote the development of Fusarium foot rot and the production of inoculum on stem bases. Intense rainfall during the period of anthesis can effectively disperse Fusarium inoculum to ears when they are most susceptible to infection (Jenkinson & Parry, 1994b). But also wind dispersed ascospores of Giberella zeae (F. graminearum) can cause ear infections (Fernando et al., 1997; Francl et al., 1999), but rain is still needed to set these spores free. Prolonged periodes of warm humid conditions are conducive to infection of cereal ears

by Fusarium spp.. Studies using F. avenaceum, F. culmorum, F. graminearum, F. poae and M. nivale, have shown that, temperatures above 15 C and wetness periods of at least 24 h are required for optimum infection of winter wheat ears (Parry et al., 1995).

Conclusion Preventative measures to reduce inoculum potential of FHB is a major option to reduce the risk of FHB epidemics. Survival of Fusarium spp. causing FHB should be limited by optimiaing crop rotation tillage and control of weeds as potential alternative hosts. Development of novel methods to enhance decomposition of cop residues of FHB infected crops may be helpful to achieve this goal.

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3. Fusarium mycotoxins in cereals: storage, processing and decontaminationA. Visconti and A. De Girolamo 3.1 IntroductionFungi of the genus Fusarium are common plant pathogens occurring world-wide in a variety of crops, although they are mainly associated with cereals. Fusarium species can produce over one hundred secondary metabolites, some of which can unfavourably affect human and animal health. The most important Fusarium mycotoxins, that can frequently occur at biologically significant concentrations in cereals, are fumonisins (mainly B1 and B2), zearalenone and trichothecenes (deoxynivalenol, nivalenol and T-2 toxin). These compounds can occur naturally in agricultural and food products, either individually or as specific clusters of two or more of them depending on the producing fungal species (or strain); they have been implicated (alone or in combination between themselves and/or with other mycotoxins) as the causative agents in a variety of animal diseases and have been associated to some human diseases. Corn is the crop most susceptible to contamination by all Fusarium mycotoxins (particularly important are fumonisins), while wheat and barley are subjected to contamination of deoxynivalenol, nivalenol and, at lesser extent, of zearalenone and T-2 toxin and related trichothecenes. Fungal infection and toxin production can occur in the field, during the storage period and post-harvest. Prevention through pre-harvest management is the main goal of agricultural and food industries for controlling mycotoxin contamination. When contamination occurs in the field and the product is to be used as human food or animal feed, the hazards associated with the toxin must be managed through harvest, storage, transportation and post-harvest. Consequently, an integrated management system, able to control every phase of production, is needed. This chapter will review the main factors responsible for Fusarium mycotoxins contamination during grain storage and decontamination strategies, including food processing, used to reduce the risk associated with the consumption of mycotoxin contaminated food or feed.

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3.2 Fungal and mycotoxin contamination in stored cropsDuring storage the cereal crop undergoes quality loss characterised by increased susceptibility to infection by fungi, insects and mites which directly or indirectly affect grain quality. Depending on the geographic origin and the storage conditions, fungi belonging to Fusarium, Penicillium and Aspergillus species, insects, yeast, and bacteria are the main responsible for spoilage of stored products. Nevertheless, fungal growth does not necessarily denote the presence of mycotoxins because not all fungal species and strains are toxigenic. Fungal invasion and mycotoxins contamination of agricultural products lead to losses in terms of quantity, market value and quality of food and feed production due to changes of colour, texture and taste (Mills, 1989; Brooker et al., 1992), development of fungal doors (Abramson, 1991; Kaminski & Wasowicz, 1991) and reduction of seed germination (Sauer, 1988). Energy and nutritional value changes in term of losses of carbohydrates, proteins, amino acids and vitamins and increases of fatty acids may also occur (Ominski et al., 1994). The species composition and the production of secondary metabolites in crop entering storage may vary depending on the presence of storage fungal infection and mycotoxins originated in pre-harvest and harvest. Interactions of several factors, such as water activity, moisture content, rapidity of drying, temperature, time, composition of the substrate, mechanical damages to the seed, oxygen and carbon dioxide availability, fungal abundance, prevalence of toxigenic strains, spore load, microbial interactions and invertebrate are responsible for fungal growth in stored crops and the eventual development of mycotoxins (Abramson, 1998). The water availability, that may be expressed as the moisture content (MC) or water activity (aw), i.e. the ratio of vapour pressure of the product to that of pure water, influences fungal growth and stability of stored products (Pitt & Hocking, 1985). A tolerance to low aw corresponds to the minimum aw at which fungal spore germination and hyphal growth can occur. Fusarium spp. are usually described as field fungi but they occasionally develop in storage when aw is higher than 0.90 (MC > 20-22%), with a minimum between 0.87 and 0.89 aw and an optimum between 0.98 and 1.00 aw. In order to obtain a good preservation of stored crops from fungal growing, the water content of grain must be removed by drying the crop until the necessary MC for safe crop storage reaches value ranging from 8% to 16.5% depending on the cereals (Bottalico, 1997; Lacey & Magan, 1991). However, too rapid heating may cause stress cracks in corn kernels increasing susceptibility to fungal invasion, while overheating may alter the relationship between water availability and water content (Lacey & Magan, 1991). Fungal contamination is also influenced by the temperature and, as with water content, each fungal species has characteristic minimum, optimum and maximum temperature requirements for growth. Some fungal species may have minimum close to or below 0C, whereas others have maximum up to 55-60C; the optimum value for the growing of 31

Fusarium spp. and production of mycotoxins on cereals is around 22-27C, except for T-2 toxin and zearalenone production that require lower temperatures, such as 2-12C and 12-15C, respectively (Bottalico, 1997). Mycotoxigenic fungi are able to grow on several substrates and the nature and amount of mycotoxin production depend on physical and chemical characteristics of the substrates. These include several parameters such as available water, mechanical resistance to packing and thermal conductivity, fat and protein content, trace mineral, amino acid and fatty acid composition. Sometimes, the presence of other micro-organisms, such as bacteria or other filamentous fungi, may alter fungal growth and mycotoxin production. The growth rate of the fungus depends on the aw, the temperature of the grain, the gaseous composition of the intergranular atmosphere and the biological properties of the competitive species (Ominski et al., 1994; Lacey & Magan, 1991). Mechanical damages from harvesting equipment together with insect, rodents or birds damage can break the outer seed coat and facilitate fungal infections. During respiration insects, together with other micro-organisms (e.g. bacteria or other fungi), can modify the environments releasing energy, which causes heating, and water, which causes moisture migration. Heating may occur in local hot spots that, having a higher water content than the bulk, may represent suitable sites for fungal infection. Certain kinds of stored-grain insects develop larvae and pupae within the infested kernel and carry numerous spores of storage fungi or through their faecal material may provide substrates for colonisation. In addition, fungi may either attract or inhibit insects or mites and may also provide food for them.

3.3 Food processing and detoxification of Fusarium mycotoxinsAlthough the prevention of mycotoxin contamination in the field is the main goal of agricultural and food industries, once the crop becomes infected under field conditions, fungal growth will continue during post-harvest phases and storage. Therefore several strategies for detoxification or decontamination of commodities containing mycotoxins have been reported and may be classified as chemical, physical, microbiological. Food processing that may involve physical and/or chemical decontamination could be considered as a strategy to destroy or redistribute mycotoxins. The ideal decontamination procedure should be easy to use, inexpensive and should not lead to the formation of compounds that are still toxic or can alters the nutritional and palatability properties of the grain or grain products. Strategies for intentional detoxification or decontamination of commodities containing mycotoxins can be classified as chemical, microbiological or 32

physical. In addition to these methods, food processing, that may involve physical and/or chemical decontamination, could destroy or reduce mycotoxins.

Food processing Processed foods are very complex systems because processing not only alters the food but also ads new ingredients and conditions, so many new interactions can occur. Fusarium mycotoxins are relatively stable under most food processing conditions and can be detected in most cereal based foods. These operations include wet and dry milling, fermentation, nixtamalization, and thermal processing such as baking, cooking, extrusion, roasting and malting. In general, factors that may influence the fate of a mycotoxin during food processing include the food matrix itself, the moisture content, whether the mycotoxin is introduced into the matrix for experimental study by natural contamination or by spiking, and its concentration. Wet and dry milling are procedures that distribute mycotoxins in the different fractions depending on the commodity and the type and level of contamination. In wet-milling of deoxynivalenol-contaminated corn much of the deoxynivalenol went into steep liquor, although measurable amounts remained in the starch (Scott, 1984). In wet-milling of zearalenone-contaminated corn it was shown that mycotoxin was mainly concentrated in the gluten (49% to 56%), followed by milling solubles (17% to 26%), while the starch fractions, corresponding to the moist products of the milling, were relatively free of zearalenone (Lopez-Garca & Park, 1998). Also for fumonisins-contaminated corn it was observed that the starch did not contain detectable fumonisin B1 residues. Fumonisin B1 remained in the fibber, gluten and germ fractions at 10-40% the level found in the starting corn (Bennett et al., 1996). Dry-milling did not significantly reduce deoxynivalenol and zearalenone levels in grains, whereas it was effective on fumonisins; in particular Katta et al. (1997) in experimentally dry-milled corn samples found the highest concentration of fumonisins in the bran and fines, whereas germ, flaking grits and grits for extrusion processing contained little or no fumonisins. It is important to realise that in some cases there may be increases in mycotoxin levels in some processed products. For example, malt may contain more zearalenone and deoxynivalenol than the unmalted barley; bran obtained after polishing barley tends to contain higher concentrations of deoxynivalenol, nivalenol and zearalenone; levels of T-2 toxin increased in corn germ after wet milling of corn; yeast doughnuts were shown to have higher concentrations of deoxynivalenol than in the flour used (Visconti et al., 2000). The traditional method used to produce masa or tortillas flour, called nixtamalization, and consisting of boiling and soaking of corn in lime water (Ca[OH]2) has been used to study the fate of fumonisin B1. Nixtamalization considerably reduced fumonisin B1 concentration in the finished products but produced hydrolysed fumonisin B1 (HFB1) which was still toxic (Murphy et al., 1996). Treatments of fumonisin B1-contaminated corn simulating modified nixtamalization (heat treatment with NaHCO3 + H2O2 alone or with 33

Ca(OH)2) gave 100% reduction of fumonisin B1 and reduced brine shrimp toxicity by ca. 40% (Park et al., 1996). A combination of heat and treatment with lime water in the process of making tortillas, reduced zearalenone by 59 to 100% and deoxynivalenol by 72 to 82% in two corn samples (Charmley & Prelusky, 1994). The fermentation processes did not destroy fumonisin, and 85% of the toxin could be recovered in all products. Consumption of these products, still containing fumonisin, by pigs, horses or other animals sensitive to relatively low levels of this toxin, could be detrimental (Lopez-Garca & Park, 1998). Other investigations showed that there was no carryover of zearalenone to distilled ethanol during fermentation, however solids containing 2 to 2.5 times the level of zearalenone in the starting product, were recovered (Lopez-Garca & Park, 1998). Fumonisins are considered to be fairly heat stable compounds. No loss of fumonisin B1 was observed when F. verticillioides culture material was boiled in water for 30 min and dried at 60 for 24h (Alberts et al., 1990) or during cooking of polenta for 20-30 min in boiling water (Pascale et al., 1995). Moreover, several investigations, focused on the effect of thermal processing on the stability of fumonisins, showed that sometimes thermally processed corn products contained lower concentrations of fumonisins than unprocessed products depending on the time and the temperature of the processes. In particular, Jackson et al. (1997) found that baking corn muffins at 175C and 200C for 20 min result in 16.3% and 27.6% reduction of fumonisin B1, respectively; no significant reduction in fumonisin B1 level was found when spiked corn masa was fried at 140 to 170C for 0 to 6 min, whereas frying chips for 15 min at 190C resulted in 67% loss of fumonisin B1. Pineiro et al. (1999) found that frying polenta or autoclaving corn meal produced reductions of fumonisin B1 of 70-80% with no conversion to the hydrolysed form. Jackson et al. (1996a,b) found that the rate and extent of fumonisins decomposition in aqueous solutions increase with processing temperature; in particular from < 27% at 125 C to > 80% at 175 C, for 60 min, depending on buffer pH. Losses of fumonisin B1 and fumonisin B2 exceeding 70% were obtained in dry corn meal heated at 190 C for 60 min and complete loss at 220C for 25 min (Scott & Lawrence, 1994). In another study it was observed that fumonisins in spiked and naturally contaminated corn meal were unstable under roasting conditions (218C for 15 min) but were stable under canning (121C up to 87 min) and baking conditions (204-232 C for 20 min), probably because the canned and baked products reached lower internal temperatures than the roasted products (Castelo et al., 1998). Extrusion processing is one of the most versatile technologies available to the food industry and it is used in the production of